Summary of the invention
[0001] The present invention relates to nitrogen fixation by leguminous plants. More specifically,
it relates to pure strains of nitrogen-fixing microorganisms that contain insert DNA
to facilitate the oxidation of H
2 produced as a byproduct of nitrogen fixation and thereby increase the amount of energy
available for the nitrogen fixation process.
[0002] To facilitate the fixation of nitrogen in leguminous plants, seeds are inoculated
with desirable strains of nitrogen-fixing microorganisms. A variety of techniques
are used, including the mixing of cultures with the soil where plants are to be grown.
More often, seeds are coated with a medium that includes the desired nitrogen-fixing
microorganism. When seeds with such a coating thereon germinate, the seedling plants
are directly exposed to the microorganism.
[0003] Nitrogenases from legume nodules and all known sources catalyze an ATP-dependent
reduction of not only N
2 to NH
4, but also protons to H
2. This H
2 production results in an inefficient use of the energy provided by the organism to
the N
2-fixing process. Most N
2-fixing, free-living microorganisms, but a minority of strains of Rhizobium, possess
the capacity for synthesis of an H
2-recycling system which oxidizes the H
2 produced during N
2 fixation, thus recapturing some of the energy expended during H
2 evolution. [Evans, H. J., Purohit, K., Cantrell, M. A., Eisbrenner, G., Russel, S.
A., Hanus, F. J., and Lepo, J. E. 1981, Current Perspectives in Nitrogen Fixation,
ed. Gibson, A. H. and Newton, W. E. (Elsevier/North Holland, New York, NY), 84-96].
[0004] Dixon [Dixon, R. O. D. 1978. Biochimie 60, 233-236] pointed out several potential
advantages of H
2-oxidizing capability to N
2-fixing organisms. Subsequent investigations have shown that an active H
2-oxidizing system in R. japonicum can increase the ATP content of bacteroid cells
and also provide a mechanism for additional protection of bacteroid nitrogenase from
O2 inactivation [Emerich, D. W., Ruiz-Argüeso, Ching, T. M., and Evans, H. J. 1979.
J. Bacteriol. 137, 153-160].
[0005] In glasshouse and field experiments, soybean plants inoculated with groups of hydrogenase
positive (Hup
+) strains of R. japonicum were reported to contain higher percentages of N in their
tissues and seeds than plants inoculated with groups of hydrogenase negative (Hup-)
strains [Albrecht, S. L., Maier, R. J., Hanus, F. J., Russell, S. A., Emerich, D.
W., and Evans, H. J. 1979, Science 203, 1255―1257], [Hanus, F. J., Albrecht, S. L.,
Zablotowicz, R. M., Emerich, D. W., Russell, S. A., and Evans, H. J. 1981, Agron.
J. 73, 368-3721. The strains of R. japonicum with highly active H
2-oxidizing capacities have been shown to grow as chemolithotrophs using H
2 and CO
2 as their sources of energy and carbon, respectively [Hanus, F. J., Maier, R. J.,
& Evans, H. J. (1979) Proc. Natl. Acad. Sci. USA 76, 1788-1792].
[0006] H
2 is oxidized by the hydrogenase enzyme and CO
2 is fixed by a RuBP carboxylase [Lepo, J. E., Hanus, F. J., and Evans, H. J. (1980)
J. Bacteriol. 141, 664―670]. Strains which possess such chemolithotrophic capacity
may have an increased chance of survival in the soil where there is a limitation of
fixed carbon substrates.
[0007] Highly active hydrogen-oxidizing systems have been found only in about 25% of R.
japonicum strains and in no strain of R. trifolii or R. meliloti [Evans, H. J., Purohit,
K., Cantrell, M. A., Eisbrenner, G., Russell, S. A., Hanus, F. J., & Lepo, J. E. (1981)
Current Perspectives in Nitrogen Fixation, eds. Gibson, A. H. & Newton, W. E., (Elsevier/North
Holland, New York, NY) 84―96]. Most strains of R. leguminosarum are Hup
- and with a few exceptions the Hup
+ strains of this species recycle only a small proportion of the H
2 produced by the nitrogenase reaction [Nelson, L. M. and Salminen, S. O. (1982) J.
Bacteriol. 151, 989―995]. It would be beneficial if Hup
- strains of any one of the several species of Rhizobium could be modified to Hup
+, e.g. by a transfer of the genes for the Hup phenotype from highly active Hup
+ strains. Beneficial effects of the transfer of genes for the Hup phenotype could
include not only the ability to utilize H
2 per se but also the ability to grow chemolithotrophically.
[0008] At least some of the determinants for the Hup phenotype have been shown to be plasmid-borne
and transmissible in Alcaligenes eutrophus [Friedrich, B., Hogrefe, C., & Schlegel,
H. G. (1981) J. Bacteriol. 147, 198-205] and in R. leguminosarum [Brewin, N. J., DeJong,
T. M., Phillips, D. A. and Johnston, A. W. B. (1980) Nature 288, 77―79]. In both R.
leguminosarum and A. eutrophus, the Hup phenotype was carried on large, naturally
occurring plasmids which must carry many other genes. The plasmid carrying Hup determinants
in R. leguminosarum also was shown to carry determinants for nodulation specificity
and for N
2 fixation. The host range for this plasmid may be limited [Brewin, N. J., DeJong,
T. M., Phillips, D. A. & Johnston, A. W. B. (1980) Nature 288, 77―79], and recipients
of this plasmid may only be able to form effective nodules on pea plants.
[0009] Techniques for measurement of H
2 uptake have been described [Hanus, F. J., Carter, K. R. & Evans, H. J. (1980) Methods
Enzymol. 69, 731-739] [Bethlenfalvay, G. J. & Phillips, D. A. (1979) Plant Physiol.
63, 816-8201. Colonies of Azotobacter chroococcum have been screened for expression
of hydrogenase activity by observations of differences in rates of reduction of methylene
blue by Hup
+ and Hup
- Azotobacter colonies [Postgate, J. R., Partridge, C. D. P., Robson, R. L., Simpson,
F. G., & Yates, M. G. (1982) J. Gen. Microbiol. 128, 905―908]. But none of these methods
relates to the isolation of Hup
+ Rhizobium colonies from a large number of colonies.
[0010] A procedure for the introduction of genetic information for hydrogen-uptake systems
into a broad host range, transmissible DNA replicon has now been developed. A definitive
method of screening large numbers of bacterial colonies for the necessary isolation
of strains carrying determinants for hydrogenase expression also has now been discovered.
Two products of these procedures, pHU1 and pHU2 DNA's, contain hydrogen-uptake genes
from Rhizobium japonicum strain 122 DES and have been transferred from Escherichia
coli to the Hup
- R. japonicum mutant strains PJ17nal and PJ18nal whereupon they converted free-living
cells of these strains, most likely by genetic complementation in the first instance
and by recombination in the second instance, to a Hup
+ phenotype. Both pHU1 and pHU2 can give bacteroids of PJ17nal, occurring in soybean
nodules, sufficient hydrogen-uptake activity to utilize all hydrogen gas produced
in the nodules. These products and other products constructed by the described procedure
may be used to introduce genetic information for a hydrogen-uptake system into strains
of Rhizobium currently lacking such systems. Expression of the introduced genetic
information in Hup- recipient Rhizobium strains will increase the amount of energy
available for nitrogen fixation.
[0011] According to one aspect of the present invention, there is provided a cosmid pHU1,
being a cosmid produced by recombinant DNA techniques and containing HU DNA, which
DNA contains hup genes or portions of hup genes, which cosmid pHU1 is present in the
culture of Escherichia coli HB101 having a deposit accession number ATCC 39195.
[0012] According to a further aspect of the present invention there is provided a cosmid
pHU2, being a cosmid produced by recombinant DNA techniques and containing HU DNA,
which cosmid pHU2 is present in the culture of Escherichia coli HB101 having deposit
accession number ATCC 39196.
[0013] Cosmid pHU1 comprises cosmid pLAFR1, being a broad host range cloning vehicle, joined
with HU DNA from Rhizobium japonicum and which upon digestion with the restriction
endonucleases listed in Table II separates into fragments of the molecular sizes identified
in Table II.
[0014] Cosmid pHU2 comprises cosmid pLAFR1, joined with HU DNA from Rhizobium japonicum
and which upon digestion with the restriction endonucleases EcoRl separates into fragments
of the molecular sizes identified in Table II.
[0015] According to a further aspect of the present invention, there is provided a replicon
which can be maintained in a Rhizobium species that is useful for nitrogen fixation
in leguminous plants, the replicon is a cloning vector joined with HU DNA necessary
for hydrogen-uptake activity, the HU DNA being of the type present in cosmid pHU1
or cosmid pHU2.
[0016] According to a further aspect of the present invention, there is provided a Escherichia
coli HB101, containing the cosmid pHU1, having the deposit accession number ATCC 39195.
[0017] According to a further aspect of the present invention, there is provided a Escherichia
coli HB101, containing the cosmid pHU2, having the deposit accession number ATCC 39196.
[0018] According to a further aspect of the present invention, there is provided a method
for producing a replicon which contains genes or portions of genes coding for hydrogen
gas uptake capacity (Hup) and which is suitable for introduction into a nitrogen-fixing
Rhizobium species, the method comprising: joining fragments of DNA which contain hup
genes or portions of hup genes, of the type present in a cosmid pHU1 or a cosmid pHU2,
to replicons which can exist in a Hup- strain of a Rhizobium species; introducing
the replicons into cells of the Hup- strain; identifying which samples of Hup- strain
have been converted to Hup
+; isolating the replicon; and introducing the replicon into other species.
[0019] According to a further aspect of the present invention, there is provided a process
for providing an active hydrogen-oxidizing system in free-living cells of a strain
of a Rhizobium species that lack such a system and that is useful for nitrogen fixation
in a leguminous plant comprising inserting a pHU1 cosmid or a pHU2 cosmid into the
cells.
[0020] Preferably this process further comprises the step of identifying a colony of Rhizobium
japonicum having hydrogen-uptake activity from a group of colonies, some of which
do not have hydrogen-uptake activity, which step comprises: replicating colonies of
the group onto a surface of a porous, sterile support material; incubating cells on
the surface under conditions that derepress their hydrogenase; treating the support
material with a methylene blue indicator solution containing inhibitors of endogenous
respiration; maintaining the support material with attached colonies in an H
2 atmosphere whereupon colonies with hydrogen uptake activity reduce the methylene
blue to its leuco form; extracting and/or cloning HU DNA from the colonies having
hydrogen up-take activity and producing a cosmid pHU1 or a cosmid pHU2, prior to inserting
the pHU1 or pHU2 cosmid into the cells.
[0021] More preferably the support material comprises filter paper. During the incubation,
the filter paper is preferably placed colony side up on plates of Repaske's medium.
The incubation may occur in a closed container wherein the atmosphere is initially
about 5% H
2, 5% CO
2, 0.7% O2, and the remainder N
2, the level of 0
2 being allowed to decrease to below 0.1% during incubation; the level of O2 thereafter
is preferably maintained at about 0.2% by additions of O2. The preferred methylene
blue indicator solution contains 200 mM iodoacetic acid, 200 mM malonic acid, 10 mM
methylene blue, 50 mM KH
2PO
4, 2.5 mM MgC1
2, and an amount of KOH sufficient to adjust the pH to 5.6.
[0022] According to a further aspect of the present invention, there is provided a process
for enhancing nitrogen fixation in leguminous plants that harbor nitrogen-fixing bacteria
normally lacking an active hydrogen-oxidizing system, the process comprising: identifying
a strain of a Rhizobium species which has desired nitrogen-fixing activity when present
in the roots of plants of a particular Leguminosae species; introducing DNA containing
hydrogen-uptake gene material of the type present in a cosmid pHU1 or a cosmid pHU2
into free-living cells of the strain so that they are converted to Hup
+ phenotype; and inoculating such plants of the particular Leguminosae species with
such cells having Hup
+ phenotype.
[0023] Preferably, the Rhizobium species is Rhizobium japonicum; and the Leguminosae species
is Glycine max.
[0024] More preferably, the DNA is introduced into the free-living cells by means of a pHU1
cosmid or a pHU2 cosmid.
[0025] According to a further aspect of the present invention there is provided a recombinant
DNA molecule comprising a first segment containing HU DNA present in cosmid pHU1 or
cosmid pHU2 flanked by second and third DNA segments that are not the DNA that flanks
the HU DNA in naturally occurring organisms.
[0026] The process of this invention employs recombinant DNA techniques in conjunction with
a screening method to allow production and isolation of recombinant replicons containing
DNA which codes for genes specific for the hydrogen-uptake phenotype. These replicons
are referred to herein as pHU DNA's. Specific examples of such replicons are the cosmids
pHU1 and pHU2 described below. Section I below is a detailed description of a process
for production and isolation of pHU DNA. Section II is a detailed description of recombinant
cosmids made by the process.
[0027] The following definitions may be useful in understanding the terminology of this
disclosure:
[0028] Replicon.-A genetic element which is capable of independent replication. This may
include a plasmid, cosmid, or bacteriophage DNA.
[0029] Hup
+.-A designation for the phenotype of any organism having the ability to show uptake
of hydrogen gas-synonymous with hydrogen gas oxidation and hydrogen uptake.
[0030] Hup-.-A designation for the phenotype of any organism which does not have the ability
to show hydrogen uptake.
hup.-A designation for a hydrogen uptake gene.
HU DNA.-DNA which contains hup genes or portions of hup genes.
pHU DNA.-A replicon produced by recombinant DNA techniques, which contains HU DNA.
I. Production and isolation of pHU DNA
[0031] An object of the present process is to isolate and produce DNA which can be used
to provide hydrogen uptake activity to Hup- Rhizobium strains and in particular DNA
which returns hydrogen uptake activity to certain specific Hup
- test strains of R. japonicum. Such DNA may come from any organism which contains
hup genes capable of either showing complementation to the Hup- test strains of R.
japonicum or showing recombination with these strains of R. japonicum, thus converting
them to a Hup
+ phenotype. The vector DNA to which the Hup gene-containing DNA (the insert DNA) is
attached may be any replicon which can be introduced and maintained in R. japonicum.
[0032] A process according to this invention includes a number of steps, including one or
more of the following:
A. Growth of bacterial strains and isolation of DNA.
B. Construction of insert DNA/vector DNA recombinants.
C. Introduction of recombinant DNA into recipient strains.
D. Identification of recipients containing pHU DNA.
E. Isolation of pHU DNA.
[0033] Step D is of particular interest in that it involves a procedure to distinguish colonies
of the Hup
+ phenotype from thousands of colonies of R. japonicum. Colonies are grown on plates
of any standard culture medium and then replicated onto a porous, sterile support
material (for example, filter paper discs) and incubated under conditions which will
derepress the hydrogen-uptake system. After an appropriate period of time, the discs
are soaked in a solution containing methylene blue and inhibitors of endogenous respiration.
The concentration of inhibitors should be sufficient to block methylene blue dye reduction
via endogenous respiration. When the discs are then incubated under a stream of H
2, only colonies with an active hydrogenase (Hup
+) will show H
2-dependent methylene blue dye reduction as evidenced by conversion of the methylene
blue in that area of the disc to a colorless form.
A. Growth of bacterial strains and isolation of DNA
[0034] Strains of E. coli, unless otherwise indicated are grown on LB medium at 32°C [Kahn,
M., et al (1979) Methods Enzymol. 68, 268-280, incorporated herein by reference].
R. japonicum are routinely cultured in broth or an agar plates using the yeast extract
mannitol medium described by Vincent [Vincent, J. M. (1970) A Manual for the Practical
Study of Root-Nodule Bacteria (Blackwell, Oxford, UK) 59―60, incorporated herein by
reference] or the hydrogen uptake medium described by Maier, et al [Maier, R. J.,
et al. (1978) Prose; Natl. Acad. Sci., USA 75,3258-32621, adjusted to pH 7.0 and modified
by adding 0.2 g KH
2PO
4 . H
20 and 0.03 g. NaH
2PO
4 .H
20 per liter instead of 0.15 g NaH
2PO
4. H
20. Strains of E. coli and R. japonicum used are listed in Table I. The cosmid, pLAFR1,
was produced by Friedman et al [Friedman, A. M. Long, S. R., Brown, S. E., Buikema,
W. J., & Ausubel, F. M. (1982) Gene 18, 289-296, incorporated herein by reference]
from the plasmid, pRK290, which was produced by G. Ditta, S. Stranfield, D. Corbin,
and D. R. Helinski [(1980), Proc. Natl. Acad. Sci., USA 77, 7347―7351, incorporated
herein by reference] and is a broad host range cloning vehicle. The nar derivatives
of SR, PJ17 and PJ18 listed in Table I are spontaneous nalidixic acid-resistant mutants
obtained by standard techniques.
[0035] Each of the writings mentioned in the above notes is incorporated herein by reference.
[0036] Large scale preparations of cosmid DNA from E. coli and R. japonicum are obtained
by the procedure of Cantrell, Hickok and Evans [(1982) Arch. Microbial 131, 102-106,
incorporated herein by reference]. For use in preparation of the clone bank, pLAFR1
DNA from E. coli is further purified by two CsCI/ethidium bromide gradient centrifugations
in 49% (w/w) CsCI and 0.35 mg/ml ethidium bromide in a buffer solution at pH 8.0 containing
50 mM Tris-HCI, 20 mM EDTA (TE buffer). The initial refractive index of the gradient
solution is 1.3910. Samples are centrifuged in a Beckman VTi65 rotor to equilibrium
(12-16 hr) at 55,000 rpm. Covalently-closed circular DNA recovered from the second
gradient centrifugation is diluted with TE buffer and pelleted by centrifugation in
a Beckman SW60 rotor. The pelleted DNA is resuspended in 0.37 ml of a buffer containing
6 mM Tris-HCI, pH 7.4, 10 mM NaCI, 0.1 mM EDTA (DNA stroage buffer), extracted repeatedly
with isoamyl alcohol and precipitated with ethanol. After drying the precipitate by
evacuation at room temperature, it is redissolved and stored in 0.25 ml DNA storage
buffer.
[0037] Small scale isolations of cosmid DNA from E. coli for use in restriction analyses
are performed by use of a cleared lysate procedure [Kahn, M., Kolter, R., Thomas,
C., Figurski, D., Meyer, R., Remaut, E., & Helinski, D. R. (1979) Methods Enzymol.
68, 271-272, incorporated herein by reference]. Total genomic DNA from 500 ml cultures
of R. japonicum grown in HUM broth medium to an optical density at 540 nm of approximately
1.0 is isolated by a previously described procedure [Haugland, R. A., & Verma, D.
P. S. (1981) J. Mol. Appl. Genet. 1, 205-217, incorporated herein by reference] with
the following modification. After the spooled, ethanol-precipitated DNA is dried,
it is redissolved in 5 ml of a pH 7.0 buffer solution containing 150 mM NaCI and 15
mM sodium citrate (1 xSSC). A 1 mg/ml solution of RNase A in the same buffer is preheated
15 minutes at 80°C and then added to the DNA solution to a final concentration of
50 µg/ml. After a 30 minute incubation at 37°C, the DNA is again spooled onto a glass
rod from an overlay of two volumes of cold ethanol and subsequently treated as referred
to above.
B. Construction of insert DNA/vector DNA recombinants
[0038] R. japonicum DNA which is to be used as the insert DNA (HU DNA) is digested with
the restriction enzyme EcoRl and size fractionated by the following procedure. R.
japonicum 122 DES total DNA (200 µg) is incubated at 37°C with 20 µl of EcoRl (Bethesda
Research Laboratories, Gaithersburg, MD, U.S.A.; 10 units/µl) for periods ranging
from 20 to 115 minutes in 100 mM Tris-HCI, pH 7.2, 50 mM NaCI, 5 mM MgCl
2 and 2 mM β - mercaptoethanol. Aliquots from each EcoRl digestion time point are subjected
to agarose gel electrophoresis by the method of Meyers et al [Meyers, J. A., Sanchez,
D., Elwell, L. P., & Falkow, S. (1976) J. Bacteriol. 127, 1529-1537, incorporated
herein by reference] with modifications of Haugland & Verma [Haugland, R. A., & Verma,
D. P. S. (1981) J. Mol. Appl. Genet, 1, 205-207, incorporated herein by reference]
to identify samples with average sizes of approximately 30 kb, 22 kb and 15 kb. Approximately
50 µg of DNA falling into each of these three classes are then pooled and extracted
first with equal volumes of phenol (equilibrated with 100 mM Tris, pH 8.5) and then
twice with double volumes of ether equilibrated as above. The DNA is then precipitated
with 2 volumes of ethanol, incubated more than 30 minutes at -20°C, pelleted by centrifugation
for 15 minutes in an Eppendorf microfuge, washed with cold 80% ethanol, recentrifuged
as above, dried under vacuum and finally redissolved in 0.7 ml of a buffer containing
20 mM Tris-HCI, 10 mM EDTA, 50 mM NaCI, pH 8.0 (sucrose gradient buffer). The sample
is heated for 10 minutes at 65°C and then layered on a sucrose gradient and centrifuged
using the conditions described by Ditta et al [Ditta, G., Stanfield, S., Corbin, D.,
and Helinski, D. R. (1980) Proc. Natl. Acad. Sci. USA 77, 7347-7351, incorporated
herein by reference]. Aliquots from 0.7 ml fractions taken from the bottom of the
gradient are then subjected to agarose gel electrophoresis as described above to identify
the fractions containing DNA in the size range of 12 to 30 kb. Fractions containing
DNA in this size range are pooled and dialyzed at 4°C against several changes of 1/10-strength
sucrose gradient buffer over a period of 24 hours. The dialyzed sample is then precipitated
with two volumes of ethanol, pelleted by centrifugation at 15,000 RPM for 20 minutes
in a Sorvall SS-34 rotor, washed with cold 80% ethanol, and recentrifuged as above,
dried under vacuum and finally redissolved in 0.4 ml of DNA storage buffer.
[0039] The pLAFR1 DNA is digested with EcoRl under the conditions described above with the
exception that EcoRl is added at a ratio of 5 units per µg of DNA, the mixture is
incubated 90 minutes at 37°C, an additional 5 units of EcoRl per µg of DNA is added,
the mixture is incubated an additional 90 minutes at 37°C, and the reaction is terminated
by adding a volume of 0.25 M Na
2EDTA (pH 8.0) equal to 6% of the reaction volume to give a final concentration of
8 mM EDTA. The reaction mixture is extracted 2 times with equal volumes of redistilled
phenol, then 3 times with equal volumes of ether, and traces of remaining ether are
then removed by evaporation with a stream of N
2.
[0040] Ligation is performed as follows: 20 µg of pooled, partially digested 12-30 kb functions
of R. japonicum DNA are added per pg of EcoRl-digested pLAFR1, the mixture is ethanol
precipitated as described above, and the precipitate is redissolved in ligation buffer
(66 mM Tris-HCI, pH 7.6, 6.6 mM MgC1
2). The resultant mixture of EcoRl-digested pLAFR1 and partially digested R. japonicum
DNA is then ligated at concentrations of 0.05 and 1.0 mg/ml, respectively, in the
presence of 700 U/ml T4 ligase (Bethesda Research Laboratories), 66 mM Tris-HCI, pH
7.6, 6.6 mM MgCl
2, 10 mM dithiothreitol and 66 µM ATP for 18 hours at 12°C.
C. Introduction of recombinant DNA into recipient strains
[0041] The recombinant DNA ligation mixture is packaged with an extract containing phage
lambda head and tail components and the resultant packaged cosmids are adsorbed onto
E. coli strain HB101.
[0042] Packaging extracts are prepared and DNA is packaged with the following modifications
of the procedure of Blattner et al. [Blattner, F. R. Blechl, A. E., Denniston-Thompson,
K., Faber, H. E., Richards, J. E., Slightom, J. L., Tucher, P. W., & Smithies, O.
(1978) Science 202, 1279-1284, incorporated herein by reference] using the lambda
lysogenic strains of E. coli BHB2688 and BHB2690 (see Table I). These lambda lysogens
are each grown as 500 ml cultures in LB medium in a 2 I flask at 32°C to approximately
3x 10
8 cells per ml. The cultures are heated in a water bath to 45°C, then maintained at
that temperature for 30 minutes with constant agitation. They are then placed on a
shaker at 37°C for 1 hour and then chilled on ice. All succeeding production of the
extracts is at 4°C.
Production of the BHB2688 extract
[0043] Cells from three 500 ml cultures of BHB2688 are each centrifuged 10 minutes at 9000
rpm in a Sorvall GSA rotor and the pellets are resuspended in a total of 3 ml of a
buffer containing 10% sucrose, 50 mM Tris-HCI, pH 7.4. A total of 150 µg of highly
purified lysozyme (Sigma) is then added in a volume of 75 µl of 0.25 M Tris-HCI, pH
7.4. The sample is mixed gently and frozen in liquid N
2. It is then thawed on ice, 400 µl of the M1 buffer of Blattner et al. is added, it
is incubated 30-45 minutes at 4°C, and centrifuged 35 minutes at 28,000 rpm in a Beckman
type 30 rotor. The BHB2688 extract consists of aliquots of the supernatant stored
at -70°C.
Production of the BHB2690 extract
[0044] Cells from one culture of 500 ml of BHB2690 are centrifuged 10 minutes at 9000 rpm
in a Sorvall GSA rotor and the pellet is resuspended in 3.1 ml of the buffer A of
Blattner et al. The suspension is sonicated as described by Blattner, et al. and centrifuged
5 minutes at 7000 rpm in a Sorvall SS-34 rotor. The BHB2690 extract consists of aliquots
of the supernatant stored at -70°C.
Packaging of DNA and transfer into E. coli
[0045] DNA packaging is initiated by addition of the following in the order indicated: 7
µl of the buffer A of Blattner et al, 3 µl of ligated DNA, 1 µl of the M1 buffer of
Blattner et al, 5 µl of BHB2690 extract and 5 µl of BHB2688 extract. After incubation
for 1 hour at 23°C, a drop of CHCI
3 is added, the mixture is serially diluted, and transduction with strain HB101 is
performed as described by Hohn [Hohn, B. (1979) Methods Enzymol. 68, 299-309, incorporated
herein by reference].
[0046] Selection for tetracycline-resistant transductance on LB plates with 20 µg/ml of
tetracycline results in the growth of a large number of colonies which constitute
a gene bank. During generation of the gene bank which lead to isolation of pHU1 and
pHU2, tetracycline resistant transductants were obtained at a frequency of 7.8x10
5 per µg of vector DNA. The resultant gene bank contained more than 40,000 clones.
Analysis of 24 of these clones chosen at random has shown that 83% contain insert
DNA with an average molecular size of 22.6 kb.
[0047] PJ17nal is an R. japonicum revertible Hup
- mutant, ATCC 39194. This biologically pure culture is available from the permanent
collection of the American Type Culture Collection, Rockville, Maryland, U.S.A. Colonies
from the clone bank were conjugated en masse with PJ17nal by use of the triparental
mating system of Ditta etal [Ditta, G., Stanfield, S., Corbin, D., & Helinski, D.
R. (1980) Proc. Natl. Acad. Sci. USA 77, 7347-7351, incorporated herein by reference].
In this procedure, cultures of E. coli containing recombinant cosmids and E. coli
containing the mobilization helper plasmid pRK2013, both grown to early log phase
(approximately 0.4 absorbance units at 600 nm) in LB medium are each concentrated
by centrifugation at 8000 rpm in a Sorvall SS-34 rotor and resuspended in 0.05 vol
of YEM medium. A culture of R. japonicum recipient cells, grown to late log phase
(approximately 0.5 absorbance units at 540 nm) in YEM medium is likewise concentrated.
Aliquots of 0.1 ml of each of these three cell suspensions (approximately 5x108 R.
japonicum cells and approximately 5x10
7 of each of the two E. coli cell types) are then spread together on a YEM medium agar
plate at pH 7.0, excess liquid is removed by evaporation under a sterile, laminar-flow
hood, and the cells are then incubated at 29°C for 3 to 4 days. After this time, the
cells are scraped off the plate and suspended in 8 ml of the mineral salts solution
used for the HUM medium containing 0.01 % Tween 80. Dilutions ranging from 10
-1 to 10-
6 are plated on either YEM or HUM medium with 100 µg/ml tetracycline and 100 pg/ml
nalidixic acid. Recipient colonies containing pHU DNA are then identified from plates
containing up to 3000 transconjugant colonies.
D. Identification of recipients containing pHU DNA
[0048] Recipient colonies which have received DNA from the clone bank (transconjugants)
are replicated onto sterile filter paper discs (Whatman 541) which are then placed
colony side up on plates of Repaske's medium [Repaske, R., & Repaske, C. (1976) Appl.
Env. Microbial, 32, 585-591; Repaske, R., & Mayer, R. (1976) Appl. Env. Microbiol.
32, 592-597, both incorporated herein by reference].
[0049] Cells on the filters are then derepressed by incubation for 4 to 5 days in an atmosphere
which initially contains by volume 5% H
2, 5% CO
2, approximately 0.7% 0
2, and the remainder N
2. 0
2 is consumed such that levels of O2 gradually decrease to less than 0.1% of the gas
volume during incubation and are subsequently maintained at about 0.2% by additions
of O
2. In cases where many Hup
+ colonies are present during the derepression period, H
2 levels also decrease as determined by gas chromatography and are subsequently maintained
at approximately 5% by additions of H
2.
[0050] Filters with depressed colonies are removed from the Repaske plates and soaked in
0.8 ml of a solution containing 200 mM iodoacetic acid (Sigman Chemical Co., St. Louis,
MO, U.S.A.), 200 mM malonic acid, 10 mM methylene blue (Nutritional Biochemical Co.),
50 mM KH
2P0
4, 2.5 mM MgCl
2 adjusted to pH 5.6 with KOH. After approximately 15 min, the plates are tilted slightly
and excess solution is removed from the sides of the filters with a Pasteur pipette.
An additional 45 min in air is allowed for the dye/inhibitor solution to equilibrate
with the colonies, and the plates with filters and attached colonies are transferred
to a Plexiglass tray (inside dimensions: 29x39x3 cm) with an outer well filled with
water. A Plexiglass cover with side walls that fit into the outer well of the tray
is used to form a partial seal against entry of air into the tray. The cover also
contains two portals to permit controlled entry and exit of cylinder gases.
[0051] N
2 is flushed through the chamber for 15 minutes to reduce the 0
2 concentration over the filters. The chamber is placed in a fume hood for safety purposes
and H
2 (passed through a catalytic O
2 purifier-Engelhard Industries, Inc.) is then introduced at a flow rate of approximately
150 ml per minute. Both gases are passed through H
20 before entering the Plexiglass chamber in order to reduce evaporation from the filters.
Colonies with H
2 uptake activity reduce methylene blue to its leuco form within 0.5 to 3 hours while
Hup
- colonies do not reduce methylene blue during periods of 20 hours and more. Hup
+ colonies are found amongst the population of Hup- recipients at frequencies of approximately
10-
2 to 10-
3. The Hup
+ colonies are then isolated from the master plates and pHU cosmid DNA is isolated
as described below.
E. Isolation of pHU DNA
[0052] Colonies showing methylene blue reduction activity in the initial screening are picked
from the original plates from which the filter replicas were taken and streaked for
single colonies on HUM medium agar plates containing 100 pg/ml each of tetracycline
and nalidixic acid. When colonies on these plates have grown to maximum size (10-15
days), the plates are replicated onto filter paper discs and the portions of the colonies
transferred are derepressed and subjected to the above-described methylene blue reduction
screening procedure for the identification of recipients containing pHU DNA. Isolated
colonies identified as having dye reducing activity in this second screening are picked
from the master plates and used to inoculate 200 ml YEM broth cultures containing
45 µg/ml tetracycline. The cultures are grown to an optical density of approximately
0.2 absorbance units at 540 nm and the cells then harvested and subjected to the plasmid
isolation procedure of Cantrell et al. [(1982) Arch. Microbial. 131, 102-1061. Partially
purified cosmid DNA obtained by this procedure is then used to transform E. coli HB101
cells by the procedure of Davis et al [Davis, R. W., Botstein, D., & Roth, J. R. (1980)
A Manual for Genetic Engineering: Advanced Bacterial Genetics (Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY) 140-141, incorporated herein by reference] with
the modification that cryogenically stored, competent E. coli cells, prepared by the
method of Morrison [Morrison, D. A. (1979) Methods Enzymol. 68, 326-331, incorporated
herein by reference] are used as recipients. The transformed E coli are selected on
LB medium with 20 µg/m) tetracycline.
[0053] The resultant transformants are used to purify pHU cosmid DNA by standard techniques.
For example, the above-described procedures for growth of bacterial strains and isolation
of DNA are used and the procedure of Guerry, LeBlanc, and Falkow [J. Bacteriol. (1973)
116, 1064―1066] followed by CsCI/EtBr gradient centrifugation is used.
II. Characteristics of pHU1 and pHU2
[0054] Two cosmids isolated by the above procedures are pHU1 and pHU2. pHU1 is obtainable
from Escherichia coli HB101 (pHU1), ATCC 39195. pHU2 is obtainable from Escherichia
coli HB101 (pHU2), ATCC 39196. These biologically pure cultures are available from
the permanent collection of the American Type Culture Collection, Rockville, MD, U.S.A.
The replicons are unique because they contain HU DNA which is attached by recombinant
DNA techniques to vector DNA. The specific vector DNA used (pLAFR1) is not itself
new. Other vector DNA may be similarly usable so long as it can be easily introduced
and maintained in R. japonicum and a bacterium such as E. coli, commonly used in genetic
laboratories. The HU DNA is the insert DNA which produces the Hup
+ character in the recipient test strains as described below. The HU DNA may come from
any organism which contains hup genes. The characteristics listed below should not
be construed as limiting, nor need the functional characteristics be specifically
associated with DNA fragments having the molecular sizes listed.
A. Physical characteristics of pHU1 and pHU2
[0055] Both the cosmids pHU1 and pHU2 are approximately 47 kb in molecular size and thus
have a molecular weight of approximately 3X10
7. The approximate molecular sizes in kilobase pairs of DNA fragments produced from
restriction endonuclease digestion of pHU1 and pHU2 with EcoRl and a combination of
EcoRl and Bglll are given in Table II. Fragments designated by asterisks in the table
occur in the cosmid vector pLAFR1 and for this reason are not fragments of HU DNA.
Results given in the table were obtained by digesting pHU1 and pHU2 for two hours
at 37° with 5.5 units EcoRl (Bethesda Research Laboratories, Inc.) per µg DNA and/or
5 units Bglll (Boerhinger Mannheim, Inc.) per µg DNA. The buffer used for all digestions
contains 50 mM Tris, pH 8.0, 10 mM MgCl
2, 50 mM NaCl and 2 mM β - mercaptoethanol. Following restriction endonuclease digestions,
the DNA's were electrophoresed in a 0.8% agarose gel made up in a buffer containing
89 mM Tris, 2.5 mM EDTA and 89 mM boric acid, pH 8.3. Additional fragments produced
in the restriction digests with sizes less than 0.4 kilobase pairs would not have
been detected in these analyses.
[0056] The DNA used as a source of HU DNA (R. japonicum 122 DES DNA) was originally digested
with the restriction enzyme EcoRl. As a result, the HU DNA present in pHU1 and pHU2
could be easily cleaved from the vector, pLAFR1, by partial digestions with EcoRl
by procedures well known in the art. Commonly used procedures would then allow joining
of the HU DNA with different replicons so that pHU insert DNA can be transferred to
many species of bacteria. Because pLAFR1 is a wide host range vector, pHU1 and pHU2
can be inserted in multiple species of bacteria whereby, for example, pHU insert DNA
can be easily maintained in E. coli, transferred from E. coli to R. japonicum, and
maintained in the R. japonicum.
B. Functional characteristics of pHU1 and pHU2
[0057] Conjugation of HB101 containing pHU1 or pHU2 with R. japonicum PJ17nal by the procedures
described above results in introduction of the cosmids into PJ17nal. Derepressed cells
of R. japonicum strain PJ17nal (see Section I, Part D above for derepression procedure)
containing pHU1 or pHU2 DNA's exhibit both methylene blue and O
2-dependent H
2 uptake activity. The aforementioned activities occur as a result of genetic complementation
of a mutated DNA sequence present within strain PJ17nal by insert DNA sequences occurring
with the HU1 or HU2 DNA's and are comparable with the activities demonstrated by derepressed
free-living cells of the Hup
+ R. japonicum strain 122 DES. Derepressed cells of PJ17na containing the vector pLAFR1
show no H
2-uptake activity.
[0058] O
2-dependent H
2-uptake activity is measured by the following method. R. japonicum cells are grown
tc an absorbance (540 nm) of 0.25 in 8 ml of YEM medium (containing 50 µg/ml of tetracycline
when strain: with plasmids are used). Cells are harvested by centrifugation and cell
pellets resuspended in 0.2 ml o HUM medium. The suspensions are spread on plates of
Repaske's medium and derepressed for 4 days under the gas mixture described for the
methylene blue colony screen. Cells are removed from the plate: and suspended in 5
ml of 50 mM KH
2P0
4 and 2.5 mM MgCl
2 buffer adjusted to pH 7.0 with KOH. The suspensions are diluted 10-fold in the above
buffer and assayed amperometrically for rates o 0
2-dependent H
2 uptake by the method of Hanus, Carter and Evans [(1980) Methods Enzymol. 69, 731―739
incorporated herein by reference].
[0059] Using the growth conditions and procedures described below, soybeans infected with
PJ17na containing pHU1 and pHU2 DNA's can form nodules which demonstrate no H
2 evolution. In a nodulatior test which was done as described below, soybean plants
infected with PJ17nal containing pHU1 or pHU2 formed nodules which demonstrated no
H
2 evolution. Acetylene reduction activities by these nodules anc those formed by strain
122 DES after similar inoculations and growth of soybeans are comparable. The pHU
insert DNA does not interfere with the ability of PJ17nal to effectively nodulate
soybean plants anc likely will not interfere with the ability of any recipient Rhizobium
species to effectively nodulate its natura plant host.
[0060] For plant tests, soybean seed [Glycine max (L.) Merr., cultivar Wilkin] are surface
disinfected by the following procedure. Seeds are soaked in 70% ethanol for 10-20
seconds, rinsed 5-8 times with sterile distilled water, incubated 3 minutes in a 2%
solution of sodium hypochlorite, then rinsed 5-8 times more with sterile distilled
water and germinated on plates of 0.8% agar, containing one-half strength N-free nutrient
solution [Harper, J. E. & Nicholas, J. C. (1976) Physiol. Plant 38, 24-28, incorporated
herein by reference] to which is added 3 mM CaS0
4. After 48 hours, seedlings are immersed for 20 minutes in day-old YEM broth cultures
(with 45 pg/ml tetracycline for transconjugants) of desired strains of R. japonicum.
The inoculated seedlings are transferred to a sterile mixture of equal parts sand
and vermiculite in 250 ml flasks. Seedlings are protected by cotton plugs and placed
in a growth cabinet (16 hr day-8 hr night cycle) at an irradiance of about 25 µE·
m
-1. sec
-1. The day and night temperatures are maintained near 28°C and 23°C, respectively.
Plants receive sufficient sterile one-half strength N-free nutrient solution [Harper,
J. E., & Nicholas, J. C. (1976) Physiol. Plant 38, 24-28, incorporated herein by reference]
to maintain a moist medium without allowing free solution in the flasks. Plants are
harvested 30 days after germination and nodules are assayed for rates of C
2H
2 reduction and H
2 evolution and for bacteroid hydrogenase activity by procedures used by Lepo et al
[Lepo, J. E., Hickok, R. E., Cantrell, M. A., Russell, S. A., & Evans, H. J. (1981)
J. Bacteriol. 146, 614―620, incorporated herein by reference].
[0061] PJ18nal is another R. japonicum revertible Hup- mutant, ATCC 39193. This biologically
pure culture is available from the permanent collection of the American Type Culture
Collection, Rockville, MD, U.S.A. Conjugation of HB101 (pHU1) or HB101 (pHU2) with
PJ18nal allows introduction of the cosmids into PJ18nal. Methylene blue-dependent
hydrogen-uptake activity is exhibited by colonies containing either pHU1 or pHU2 at
frequencies of approximately 1-3x103.
[0062] These frequencies of appearance of Hup
+ colonies are calculated by dividing the number of Hup
+ tetracycline resistant colonies by the total number of tetracycline resistant colonies.
[0063] The strains PJ17nal and pJ18nal lack the ability to grow chemolithotrophically only
because they are Hup- due to a single point mutation. The strain from which they were
derived, SR, had the ability to grow chemolithotrophically. A number of PJ17nal transconjugants
which were coverted to Hup
+ were identified by their ability to grow chemolithotrophically after introduction
of pHU cosmids. The cosmids pHU1 and pHU2 were not analyzed in this manner, but since
they convert PJ17nal to a Hup
+ character, they should also convert PJ17nal into a chemolithotroph.
[0064] While we have described and given examples of preferred embodiments of our inventions,
it will be apparent to those skilled in the art that changes and modifications may
be made without departing from our inventions in their broader aspects. We therefore
intend the appended claims to cover all such changes and modifications as fall within
the true spirit and scope of our inventions.
[0065] The features disclosed in the foregoing description, in the following claims and/or
in the accompanying drawings may, both separately and in any combination thereof,
be material for realising the invention in diverse forms thereof.
1. Cosmid pHU1, welches ein Cosmid ist, das durch rekombinante DNA-Techniken hergestellt
ist und HU-DNA enthält, die hup-Gene oder Teile von hup-Genen enthält, wobei das Cosmid
pHU1 in der Kultur von Escherichia coli HB101 mit einer Hinterlegungszugriffsnummer
ATCC 39195 vorhanden ist.
2. Cosmid pHU2, welches ein Cosmid ist, das durch rekombinante DNA-Techniken hergestellt
ist und HU-DNA enthält, wie in Anspruch 1 definiert, wobei das Cosmid pHU2 in der
Kultur von Escherichia coli HB101 mit der Hinterlegungszugriffsnummer ATCC 39196 vorhanden
ist.
3. Replikon, das in einer Rhizobium-Spezies gehalten werden kann, die für die Stickstoff-Fixierung
in Leguminosen nützlich ist, wobei das Replikon ein Klonierungsvektor ist, der mit
HU-DNA, wie in Anspruch 1 definiert, verbunden ist, die für Wasserstoffaufnahmeaktivität
notwendig ist, wobei die HU-DNA diejenige HU-DNA ist, die in Cosmid pHU1, wie in Anspruch
1 definiert, oder in Cosmid pHU2, wie in Anspruch 2 definiert, vorhanden ist.
4. Escherichia coli HB101, welches das Cosmid pHU1, wie in Anspruch 1 definiert, enthält,
mit der Hinterlegungszugriffsnummer ATCC 39195.
5. Biologisch reine Kultur des Escherichia coli HB101 gemäß Anspruch 4.
6. Escherichia coli HB101, welches das Cosmid pHU2, wie in Anspruch 2 definiert, enthält,
mit der Hinterlegungszugriffsnummer ATCC 39196.
7. Biologisch reine Kultur des Escherichia coli HB101 gemäß Anspruch 6.
8. Verfahren zum Herstellen eines Replikons, das Gene oder Teile von Genen enthält,
die für Wasserstoffgasaufnahmevermögen (Hup) kodieren, und das zur Einführung in eine
stickstoff-fixierende Rhizobium-Spezies geeignet ist, wobei das Verfahren folgendes
umfaßt: Verbinden von DNA-Fragmenten, die hup-Gene oder Teile von hup-Genen enthalten,
von der Art, wie sie in einem Cosmid pHU1, wie in Anspruch 1 definiert, oder in einem
Cosmid pHU2, wie in Anspruch 2 definiert, vorhanden sind, mit Replikons, die in einem
Hup--Stamm einer Rhizobium-Spezies existieren können; Einführen der Replikons in Zellen
des Hup--Stammes; Identifizieren, welche Proben des Hup--Stamms in Hup+ überführt worden sind; und Isolieren der Replikons.
9. Verfahren zum Bereitstellen eines aktiven wasserstoffoxidierenden Systems in freilebenden
Zellen eines Stamms einer Rhizobium-Spezies, der ein solches System fehlt und die
für die Stickstoff-Fixierung in einer Leguminose nützlich ist, welches das Einführen
eines pHU1-Cosmids, wie in Anspruch 1 definiert, oder eines pHU2-Cosmids, wie in Anspruch
2 definiert, in die Zellen umfaßt.
10. Verfahren nach Anspruch 9, wobei das Verfahren weiterhin den Schritt des Identifizierens
einer Kolonie von Rhizobium japonicum mit Wasserstoffaufnahmeaktivität aus einer Gruppe
von Kolonien umfaßt, von denen einige keine Wasserstoffaufnahmeaktivität aufweisen,
wobei dieser Schritt folgendes umfaßt: Replizieren von Kolonien der Gruppe auf eine
Oberfläche eines porösen, sterilen Trägermaterials; Inkubieren von Zellen auf der
Oberfläche unter Bedingungen, die ihre Hydrogenase dereprimieren; Behandeln des Trägermaterials
mit einer Methylenblau-Indikatorlösung, die Inhibitoren der endogenen Atmung enthält;
Halten des Trägermaterials mit anhängenden Kolonien in einer H2-Atmosphäre, woraufhin Kolonien mit Wasserstoffaufnahmeaktivität das Methylenblau
zu seiner Leuko-Form reduzieren; Extrahieren und/oder Klonieren von Hu-DNA, wie in
Anspruch 1 definiert, aus Kolonien mit Wasserstoffaufnahmeaktivität und Herstellen
eines Cosmids pHU1, wie in Anspruch 1 definiert, oder eines Cosmids pHU2, wie in Anspruch
2 definiert, vor dem Einführen des pHU1- oder pHU2-Cosmids in die Zellen.
11. Verfahren nach Anspruch 10, wobei: das Trägermaterial Filterpapier umfaßt; während
des Inkubierens das Filterpapier mit der Kolonieseite nach oben auf Platten von Repaske-Medium
gelegt werden; des Inkubieren in einem geschlossenen Behälter abläuft, wobei die Atmosphäre
anfänglich etwa 5% H2, 5% C02, 0,7% 02 und der Rest N2 ist, wobei man zuläßt, daß der O2-Gehaltwährend des Inkubierens bis unter 0,1% absinkt; der 02-Gehalt danach durch Zugaben von O2 bei etwa 0,2% gehalten wird; die Methylenblau-Indikatorlösung 200 mM Jodessigsäure,
200 mM Malonsäure, 10 mM Methylenblau, 50 mM KH2P04, 2,5 mM MgC12 und eine Menge an KOH enthält, die ausreicht, um den pH auf 5,6 einzustellen.
12. Verfahren zum Steigern der Stickstoff-Fixierung in Leguminosen, die stickstoff-fixierende
Baktieren beherbergen, welches normalerweise ein aktives wasserstoffoxidierendes System
fehlt, wobei das Verfahren folgendes umfaßt: Identifizieren eines Stamms einer Rhizobium-Spezies,
welches die gewünschte stickstoff-fixierende Aktivtät aufweist, wenn sie in den Wurzeln
von Pflanzen einer bestimmten Leguminosen-Spezies vorhanden ist; Einführen von DNA,
die wasserstoffaufnehmendes Genmaterial der Art enthält, die in einem Cosmid pHU1,
wie in Anspruch 1 definiert, oder einem Cosmid pHU2, wie in Anspruch 2 definiert,
vorhanden ist, in freilebenden Zellen des Stamms, so daß sie in den Hup+-Phänotyp überführt werden; und Beimpfen solcher Pflanzen der besonderen Leguminosen-Spezies
mit solchen Zellen mit Hup+-Phänotyp.
13. Verfahren nach Anspruch 12, wobei: die Rhizobium-Spezies Rhizobium japonicum ist;
und die Leguminosen Spezies Glycine max ist.
14. Verfahren nach Anspruch 12, welches das Einführen eines pHU1-Cosmids, wie in Anspruch
1 definiert, oder eines pHU2-Cosmids, wie in Anspruch 2 definiert, in die freilebenden
Zellen umfaßt.
15. Rekombinantes DNA-Molekül, welches ein erstes Segment umfaßt, das HU-DNA, wie
in Anspruch 1 definiert, enthält, das in Cosmid pHU1, wie in Anspruch 1 definiert,
oder Cosmid pHU2, wie in Anspruch 2 definiert, vorhanden ist, flankiert von zweiten
und dritten DNA-Segmenten, die nicht die DNA sind, welche die natürlich vorkommende
HU-DNA flankieren.